EP2305306B1 - Inhibiteur de neuro-invasion - Google Patents

Inhibiteur de neuro-invasion Download PDF

Info

Publication number
EP2305306B1
EP2305306B1 EP09758415.5A EP09758415A EP2305306B1 EP 2305306 B1 EP2305306 B1 EP 2305306B1 EP 09758415 A EP09758415 A EP 09758415A EP 2305306 B1 EP2305306 B1 EP 2305306B1
Authority
EP
European Patent Office
Prior art keywords
antibody
receptor
cells
antibodies
human
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP09758415.5A
Other languages
German (de)
English (en)
Other versions
EP2305306A1 (fr
EP2305306A4 (fr
Inventor
Shuichi Mitsunaga
Atsushi Ochiai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chugai Pharmaceutical Co Ltd
National Cancer Center Japan
Original Assignee
Chugai Pharmaceutical Co Ltd
National Cancer Center Japan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chugai Pharmaceutical Co Ltd, National Cancer Center Japan filed Critical Chugai Pharmaceutical Co Ltd
Publication of EP2305306A1 publication Critical patent/EP2305306A1/fr
Publication of EP2305306A4 publication Critical patent/EP2305306A4/fr
Application granted granted Critical
Publication of EP2305306B1 publication Critical patent/EP2305306B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001119—Receptors for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to neural invasion-suppressing agents. Specifically, the present invention relates to neural invasion-suppressing agents that comprise an anti-interleukin 6 (IL-6) receptor antibody as an active ingredient.
  • IL-6 anti-interleukin 6
  • IL-6 is a cytokine also called B-cell stimulating factor 2 (BSF2) or interferon ⁇ 2.
  • BSF2 B-cell stimulating factor 2
  • IL-6 was discovered as a differentiation factor involved in the activation of B-cell lymphocytes (Non-Patent Document 1), and was later revealed to be a multifunctional cytokine that influences the function of various cells (Non-Patent Document 2). It has been reported to induce maturation of T lymphocyte cells (Non-Patent Document 3).
  • IL-6 transmits its biological activity via two kinds of proteins on the cell.
  • the first is the IL-6 receptor, which is a ligand-binding protein to which IL-6 binds, with a molecular weight of about 80 kDa (Non-Patent Documents 4 and 5).
  • the IL-6 receptor is present in a membrane-bound form that penetrates the cell membrane. It is expressed on the cell membrane, and also as a soluble IL-6 receptor, which mainly consists of the extracellular region of the membrane-bound form.
  • the other kind of protein is the membrane protein gp130, which has a molecular weight of about 130 kDa and is involved in non-ligand binding signal transduction.
  • the biological activity of IL-6 is transmitted into the cell through formation of an IL-6/IL-6 receptor complex by IL-6 and I1-6 receptor followed by binding of the complex with gp130 (Non-Patent Document 6).
  • pancreatic cancer Even today, many pancreatic cancer cases are diagnosed at the advanced and unresectable stage. Furthermore, even in cases that have undergone resection, which is the only way a cure can be expected, pancreatic cancer often recurs early after surgery. Meanwhile, chemotherapy is indicated for unresectable cases with good performance status (PS) and major organ function. However, although chemotherapy is currently a standard therapy, its therapeutic effect is insufficient. For example, even when gemcitabine hydrochloride regarded as the first drug of choice is used, the efficacy rate of palliative effects is 23.8%, the median survival time is 5.7 months, and the one-year survival rate is 18% (foreign phase III clinical trial data). In Japan, 20,000 people are diagnosed as having pancreatic cancer each year, and 22,260 people died of this disease in 2004 (statistics reported by the Ministry of Health, Labour and Welfare), and pancreatic cancer is the fifth cause of cancer death.
  • PS performance status
  • pancreatic cancer Even today, many pancreatic cancer cases are diagnosed at the advanced and unre
  • Neural invasion is one of the modes of invasion characteristic of pancreatic cancer.
  • the present inventors have revealed that: neural invasion is found in almost 100% of pancreatic cancer cases; it is an important prognostic factor; and it causes functional abnormalities of hepatocytes, and thus is correlated with cachectic symptoms such as anemia, impaired performance status (PS), and undernutrition.
  • neural invasion is thought to be the cause of cancer pain and the like, and there are some reports describing that symptoms were relieved to some extent by irradiating predominant sites of neural invasion, or by excising the nerve upstream of the sites.
  • the mechanisms of neural invasion and onset of symptoms caused by neural invasion are poorly understood, and thus there is currently little information on control of neural invasion and symptoms caused by neural invasion.
  • Neural invasion is commonly found regardless of cancer types, and it has been reported as a prognostic factor of prostatic cancer, stomach cancer, and head and neck cancer.
  • an objective of the present invention is to provide novel neural invasion-suppressing agents. Furthermore, the present invention provides novel agents for treating pancreatic cancer.
  • pancreatic cancer a model for neural invasion by pancreatic cancer.
  • the present invention was completed.
  • the present inventors also demonstrated that an IL-6 receptor is expressed in human pancreatic cancer cell lines, and that IL-6 enhances the chemotactic and migratory activities and intracellular signaling of pancreatic cancer cells, and thus pancreatic cancer can be treated by inhibiting IL-6.
  • the present invention provides [1] to [9] below.
  • IL-6 inhibitor refers to a substance that blocks IL-6 signaling and inhibits the biological activity of IL-6.
  • the IL-6 inhibitors include, for example, substances that bind to IL-6, substances that bind to an IL-6 receptor, and substances that bind to gp130.
  • the IL-6 inhibitors also include substances that inhibit phosphorylation of STAT3 which is important for the intracellular signaling of IL-6, such as AG490.
  • the IL-6 inhibitors include, but are not particularly limited to, anti-IL-6 antibodies, anti-IL-6 receptor antibodies, anti-gp130 antibodies, IL-6 variants, soluble IL-6 receptor variants, partial peptides of IL-6, partial peptides of an IL-6 receptor, and low-molecular-weight compounds having an activity equivalent thereto.
  • the IL-6 inhibitors include IL-6 receptor inhibitors, in particular, anti-IL-6 receptor antibodies.
  • antibodies used herein is not particularly limited; however, the antibodies are preferably derived from mammals, and more preferably from human.
  • An antibody of the present disclosure can be prepared as a polyclonal or monoclonal antibody using known methods.
  • monoclonal antibodies derived from mammals are preferably used in the present invention.
  • Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by hosts transformed with an expression vector carrying an antibody gene using genetic engineering techniques. Typically, these antibodies block transmission of the biological activity of IL-6 into cells by binding to IL-6, an IL-6 receptor, gp130, or the like.
  • monoclonal antibody-producing hybridomas can be prepared using known techniques as follows. Specifically, immunization is carried out by a conventional immunization method using as a sensitizing antigen an IL-6 receptor, IL-6, gp130, or such. The resulting immune cells are fused with known parental cells by a conventional cell fusion method. Then, monoclonal antibody-producing cells are screened using a conventional screening method.
  • monoclonal antibodies can be produced as follows.
  • a human IL-6 receptor or mouse IL-6 receptor for use as a sensitizing antigen for obtaining antibodies can be obtained by using the IL-6 receptor genes and/or amino acid sequences disclosed in European Patent Application Publication No. EP 325474 and Japanese Patent Application Kokai Publication No. ( JP-A) Hei 3-155795 , respectively.
  • IL-6 receptor proteins There are two kinds of IL-6 receptor proteins: one expressed on the cell membrane and the other separated from the cell membrane (soluble IL-6 receptors) ( Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676 ).
  • the soluble IL-6 receptor essentially consists of the extracellular region of the cell membrane-bound IL-6 receptor, and differs from the membrane-bound IL-6 receptor in that it lacks the transmembrane region or both the transmembrane and intracellular regions.
  • Any IL-6 receptor may be employed as an IL-6 receptor protein, so long as it can be used as a sensitizing antigen for producing an anti-IL-6 receptor antibody herein.
  • the desired IL-6 receptor protein is purified from the inside of the host cell or from the culture supernatant using a known method.
  • This purified IL-6 receptor protein may be used as a sensitizing antigen.
  • a cell expressing the IL-6 receptor or a fusion protein of the IL-6 receptor protein and another protein may be used as a sensitizing antigen.
  • human IL-6 when IL-6 is used as a sensitizing antigen for preparation of antibodies, human IL-6 can be obtained by using the gene and/or amino acid sequences of IL-6 disclosed in Eur. J. Biochem (1987) 168, 543-550 , J. Immunol. (1988) 140, 1534-1541 , or Agr. Biol. Chem. (1990) 54, 2685-2688 .
  • the gene and/or amino acid sequences of gp130 disclosed in European Patent Application Publication No. EP 411946 can be used.
  • Mammals to be immunized with a sensitizing antigen are not particularly limited, but are preferably selected considering compatibility with the parent cell used for cell fusion. Generally, rodents such as mice, rats, and hamsters are used.
  • Animals are immunized with sensitizing antigens according to known methods.
  • animals are immunized by intraperitoneal or subcutaneous injection of a sensitizing antigen.
  • the sensitizing antigen is preferably diluted or suspended in an appropriate amount of phosphate-buffered saline (PBS), physiological saline or such, mixed with an appropriate amount of a general adjuvant (e . g ., Freund's complete adjuvant), emulsified, and then administered to a mammal several times, every four to 21 days.
  • PBS phosphate-buffered saline
  • a general adjuvant e . g ., Freund's complete adjuvant
  • an appropriate carrier may be used for immunization with a sensitizing antigen.
  • immune cells are obtained from the mammal for cell fusion.
  • Preferred immune cells for cell fusion include, in particular, spleen cells.
  • the mammalian myeloma cells used as parent cells, i.e. as partner cells to be fused with the above immune cells include various known cell strains, for example, P3X63Ag8.653 ( Kearney, J. F. et al., J. Immunol (1979) 123, 1548-1550 ), P3X63Ag8U.1 ( Current Topics in Microbiology and Immunology (1978) 81, 1-7 ), NS-1 ( Kohler, G. and Milstein, C., Eur. J. Immunol. (1976) 6, 511-519 ), MPC-11 ( Margulies, D. H. et al., Cell (1976) 8, 405-415 ), SP2/0 ( Shulman, M.
  • P3X63Ag8.653 Kearney, J. F. et al., J. Immunol (1979) 123, 1548-1550
  • P3X63Ag8U.1 Current Topics in Microbiology and Immunology
  • cell fusion of the aforementioned immune cells and myeloma cells can be performed using known methods, for example, the method of Milstein et al. (Kohler, G and Milstein, C., Methods Enzymol. (1981) 73, 3-46 ), and such.
  • the aforementioned cell fusion is achieved in general nutrient culture medium in the presence of a cell fusion enhancing agent.
  • a cell fusion enhancing agent for example, polyethylene glycol (PEG), Sendai virus (HVJ), and such are used as fusion enhancing agents.
  • auxiliary agents such as dimethyl sulfoxide may be added depending on the need.
  • the ratio of immune cells to myeloma cells used is preferably, for example, 1 to 10 immune cells for each myeloma cell.
  • the culture medium used for the aforementioned cell fusion is, for example, the RPMI 1640 or MEM culture medium, which are suitable for proliferation of the aforementioned myeloma cells.
  • a general culture medium used for culturing this type of cell can also be used.
  • serum supplements such as fetal calf serum (FCS) can be used in combination.
  • the fusion cells (hybridomas) of interest are formed by mixing predetermined amounts of an aforementioned immune cell and myeloma cell in an aforementioned culture medium, and then adding and mixing a concentration of 30% to 60% (w/v) PEG solution (e.g ., a PEG solution with a mean molecular weight of about 1,000 to 6,000) pre-heated to about 37°C. Then, cell fusion agents and such that are unsuitable for the growth of hybridomas can be removed by repeatedly adding an appropriate culture medium and then removing the supernatant by centrifugation.
  • a concentration of 30% to 60% (w/v) PEG solution e.g ., a PEG solution with a mean molecular weight of about 1,000 to 6,000
  • the above hybridomas are selected by culturing cells in a general selection culture medium, for example, HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture in HAT culture medium is continued for a sufficient period, generally several days to several weeks, to kill cells other than the hybridomas of interest (unfused cells). Then, a standard limited dilution method is performed to screen and clone hybridomas that produce an antibody of interest.
  • HAT culture medium a culture medium containing hypoxanthine, aminopterin, and thymidine.
  • desired human antibodies with the activity of binding to a desired antigen or antigen-expressing cell can be obtained by sensitizing a human lymphocyte with a desired antigen protein or antigen-expressing cell in vitro, and fusing the sensitized B lymphocyte with a human myeloma cell (e.g., U266) (see, Japanese Patent Application Kokoku Publication No. ( JP-B) Hei 1-59878 (examined, approved Japanese patent application published for opposition)).
  • JP-B Japanese Patent Application Kokoku Publication No.
  • a desired human antibody can be obtained by administering an antigen or antigen-expressing cell to a transgenic animal that has a repertoire of human antibody genes, and then following the aforementioned method (see, International Patent Application Publication Nos. WHO 93/12227 , WO 92/03918 , WO 94/02602 , WO 94/25585 , WO 96/34096 , and WO 96/33735 ).
  • the thus-prepared hybridomas which produce monoclonal antibodies can be subcultured in a conventional culture medium and stored in liquid nitrogen for a long period.
  • the following methods may be employed: (1) methods where the hybridomas are cultured according to conventional methods and the antibodies are obtained as a culture supernatant; (2) methods where the hybridomas are proliferated by administering them to a compatible mammal and the antibodies are obtained as ascites; and so on.
  • the former method is preferred for obtaining antibodies with high purity, and the latter is preferred for large-scale antibody production.
  • anti-IL-6 receptor antibody-producing hybridomas can be prepared by the method disclosed in JP-A (Kokai) Hei 3-139293 .
  • Such hybridomas can be prepared by injecting a PM-1 antibody-producing hybridoma into the abdominal cavity of a BALB/c mouse, obtaining ascites, and then purifying a PM-1 antibody from the ascites; or by culturing the hybridoma in an appropriate medium (e.g ., RPMI 1640 medium containing 10% fetal bovine serum, and 5% BM-Condimed H1 (Boehringer Mannheim); hybridoma SFM medium (GIBCO-BRL); PFHM-II medium (GIBCO-BRL), etc .) and then obtaining PM-1 antibody from the culture supernatant.
  • an appropriate medium e.g ., RPMI 1640 medium containing 10% fetal bovine serum, and 5% BM-Condimed H1 (Boehringer Mannheim)
  • Recombinant antibodies can be used as the monoclonal antibodies described herein, wherein the antibodies are produced using genetic recombination techniques by cloning an antibody gene from a hybridoma, inserting the gene into an appropriate vector, and then introducing the vector into a host (see, for example, Borrebaeck, C. A. K. and Larrick, J. W., Therapeutic Monoclonal Antibodies, published in the United Kingdom by Macmillan Publishers Ltd, 1990 ).
  • mRNAs coding for antibody variable (V) regions are isolated from cells that produce antibodies of interest, such as hybridomas.
  • mRNAs can be isolated by preparing total RNAs according to known methods, such as the guanidine ultracentrifugation method ( Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299 ) and the AGPC method ( Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159 ), and preparing mRNAs using the an mRNA Purification Kit (Pharmacia) and such.
  • mRNAs can be directly prepared using a QuickPrep mRNA Purification Kit (Pharmacia).
  • cDNAs of the antibody V regions are synthesized from the obtained mRNAs using reverse transcriptase.
  • cDNAs may be synthesized using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit and so on.
  • the 5'-RACE method Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002 ; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932 ) using 5'-Ampli FINDER RACE Kit (Clontech) and PCR may be employed.
  • a DNA fragment of interest is purified from the obtained PCR products and then ligated with a vector DNA. Then, a recombinant vector is prepared using the above DNA and introduced into Escherichia coli or such, and then its colonies are selected to prepare a desired recombinant vector.
  • the nucleotide sequence of the DNA of interest is confirmed by, for example, the dideoxy method.
  • the DNA is ligated with a DNA that encodes a desired antibody constant region (C region), and inserted into an expression vector.
  • a DNA encoding an antibody V region may be inserted into an expression vector comprising a DNA of an antibody C region.
  • an antibody gene is inserted into an expression vector such that it is expressed under the control of an expression regulating region, for example, an enhancer and promoter. Then, the antibody can be expressed by transforming a host cell with this expression vector.
  • artificially modified genetic recombinant antibodies for example, chimeric antibodies, humanized antibodies, or such, can be used.
  • modified antibodies can be prepared using known methods.
  • a chimeric antibody can be obtained by ligating a DNA encoding an antibody V region, obtained as above, with a DNA encoding a human antibody C region, then inserting the DNA into an expression vector and introducing it into a host for production (see, European Patent Application Publication No. EP 125023 ; International Patent Application Publication No. WO 92/19759 ). This known method can be used to obtain chimeric antibodies useful for the present invention.
  • Humanized antibodies are also referred to as reshaped human antibodies, and are antibodies wherein the complementarity determining regions (CDRs) of an antibody from a mammal other than human (e.g ., a mouse antibody) are transferred into the CDRs of human antibodies.
  • CDRs complementarity determining regions
  • General methods for this gene recombination are also known (see, European Patent Application Publication No. EP 125023 , International Patent Application Publication No. WO 92/19759 ).
  • DNA sequences designed such that the CDRs of a mouse antibody are ligated with the framework regions (FRs) of a human antibody are synthesized by PCR from several oligonucleotides produced to contain overlapping portions at their termini.
  • the obtained DNA is ligated with a human antibody C region-encoding DNA and then inserted into an expression vector.
  • the expression vector is introduced into a host to produce the humanized antibody (see, European Patent Application Publication No. EP 239400 , International Patent Application Publication No. WO 92/19759 ).
  • the human antibody FRs to be ligated via the CDRs are selected so that the CDRs form suitable antigen binding sites.
  • the amino acid(s) within the FRs of the antibody variable regions may be substituted as necessary so that the CDRs of the reshaped human antibody form an appropriate antigen binding site ( Sato, K. et al., Cancer Res. (1993) 53, 851-856 ).
  • Human antibody heavy chain C regions are generally used for the chimeric and humanized antibodies, and include C ⁇ etc. For example, C ⁇ 1, C ⁇ 2, C ⁇ 3, or C ⁇ 4 may be used. Human antibody light chain C regions include, for example, C ⁇ and C ⁇ . Furthermore, to improve the stability of the antibodies or their production, the human antibody C regions may be modified.
  • Chimeric antibodies consist of the variable region of an antibody derived from a non-human mammal and the constant region of an antibody derived from a human; humanized antibodies consist of the CDRs of an antibody derived from a non-human mammal and the framework regions and constant regions derived from a human antibody. They have reduced antigenicity in the human body, and are thus useful as antibodies for use as pharmaceuticals.
  • variable regions of human antibodies can be expressed on phage surfaces as single chain antibodies (scFv) by using the phage display method, and antigen-binding phages can then be selected.
  • scFv single chain antibodies
  • DNA sequences coding for the human antibody variable regions that bind to the antigen can be determined. Once the DNA sequence of an scFv that binds to the antigen is revealed, an appropriate expression vector comprising the sequence can be constructed to obtain a human antibody.
  • the antibody genes constructed above can be expressed according to conventional methods.
  • the antibody gene can be expressed using a DNA in which the antibody gene to be expressed is functionally ligated to a useful commonly used promoter and a poly A signal downstream of the antibody gene, or a vector comprising the DNA.
  • a promoter/enhancer include the human cytomegalovirus immediate early promoter/enhancer.
  • retroviruses polyoma viruses
  • adenoviruses adenoviruses
  • SV40 simian virus 40
  • HEF1 ⁇ human elongation factor 1 ⁇
  • the expression can be easily performed by following the method by Mulligan et al. ( Mulligan, R. C. et al., Nature (1979) 277, 108-114 ).
  • the method by Mizushima et al. Mizushima, S. and Nagata S., Nucleic Acids Res. (1990) 18, 5322 ) can be easily used.
  • Production systems using prokaryotic cells include those using bacterial cells.
  • Known bacterial cells include E. coli and Bacillus subtilis .
  • an antibody gene can be expressed by functionally ligating a conventional promoter, a signal sequence for antibody secretion, and the antibody gene to be expressed.
  • the promoter include a lacZ promoter, araB promoter and such.
  • genes can be expressed according to the method of Ward et al. ( Ward, E. S. et al., Nature (1989) 341, 544-546 ; Ward, E. S. et al., FASEB J. (1992) 6, 2422-2427 ); and the araB promoter may be used according to the method of Better et al. ( Better, M. et al., Science (1938) 240, 1041-1043 ).
  • the pel B signal sequence Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379-4383 ) may be used as a signal sequence for antibody secretion.
  • the antibodies produced into the periplasm are isolated, and then used after appropriately refolding the antibody structure (see, for example, WO 96/30394 ).
  • the expression vector may comprise the aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, or such as a selection marker.
  • APH aminoglycoside phosphotransferase
  • TK thymidine kinase
  • Ecogpt E. coli xanthine-guanine phosphoribosyltransferase
  • dhfr dihydrofolate reductase
  • the production systems for antibody preparation include in vitro and in vivo production systems.
  • In vitro production systems include those using eukaryotic cells or prokaryotic cells.
  • the production systems include those using animal cells, plant cells, or fungal cells.
  • animal cells include (1) Mammalian cells, for example, CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero, and such; (2) amphibian cells, for example, Xenopus oocyte; and (3) insect cells, for example, sf9, sf21, Tn5, and such.
  • Known plant cells include cells derived from Nicotiana tabacum, which may be cultured as a callus.
  • Known fungal cells include yeasts such as Saccharomyces ( e.g ., S. cerevisiae ), mold fungi such as Aspergillus ( e.g ., A. niger ), and such.
  • Antibodies can be obtained by using transformation to introduce an antibody gene of interest into these cells, and then culturing the transformed cells in vitro. Cultures are conducted according to known methods. For example, DMEM, MEM, RPMI 1640, IMDM may be used as the culture medium, and serum supplements such as FCS may be used in combination. Further, cells introduced with antibody genes may be transferred into the abdominal cavity or such of an animal to produce the antibodies in vivo.
  • in vivo production systems include those using animals or plants.
  • Production systems using animals include those that use mammals or insects.
  • Mammals that can be used include goats, pigs, sheep, mice, bovines and such ( Vicki Glaser, SPECTRUM Biotechnology Applications, 1993 ). Further, insects that can be used include silkworms. When using plants, tobacco may be used, for example.
  • an antibody gene is introduced into these animals or plants, the antibody is produced in the body of the animals or plants, and this antibody is then recovered.
  • an antibody gene can be prepared as a fusion gene by inserting it into the middle of a gene encoding a protein such as goat ⁇ casein, which is uniquely produced into milk. DNA fragments comprising the fusion gene, which includes the antibody gene, are injected into goat embryos, and the embryos are introduced into female goats.
  • the desired antibody is obtained from milk produced by the transgenic animals born to the goats that received the embryos, or produced from progenies of these animals.
  • the transgenic goats can be given hormones to increase the volume of milk containing the desired antibody that they produce ( Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702 ).
  • the silkworms When silkworms are used, the silkworms are infected with a baculovirus inserted with a desired antibody gene, and the desired antibody is obtained from the body fluids of these silkworm ( Maeda, S. et al., Nature (1985) 315, 592-594 ).
  • the desired antibody gene is inserted into a plant expression vector (e . g ., pMON530) and the vector is introduced into bacteria such as Agrobacterium tumefaciens .
  • This bacterium is used to infect tobacco ( e.g ., Nicotiana tabacum ) such that desired antibodies can be obtained from the leaves of this tobacco ( Julian, K. -C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138 ).
  • DNAs encoding an antibody heavy chain (H chain) and light chain (L chain) may be inserted into separate expression vectors and a host is then co-transformed with the vectors.
  • the DNAs may be inserted into a single expression vector for transforming a host (see International Patent Application Publication No. WO 94/11523 ).
  • antibody fragments may be antibody fragments or modified products thereof, so long as they can be suitably used : herein.
  • antibody fragments include Fab, F(ab')2, Fv, and single chain Fv (scFv), in which the Fvs of the H and L chains are linked via an appropriate linker.
  • the antibody fragments are produced by treating antibodies with enzymes, for example, papain or pepsin, or alternatively, genes encoding these fragments are constructed, introduced into expression vectors, and these are expressed in appropriate host cells (see, for example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976 ; Better, M. & Horwitz, A. H., Methods in Enzymology (1989) 178, 476-496 ; Plueckthun, A. & Skerra, A., Methods in Enzymology (1989) 178, 497-515 ; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663 ; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-666 ; Bird, R. E. et al., TIBTECH (1991) 9, 132-137 ).
  • enzymes for example, papain or pepsin
  • An scFv can be obtained by linking the H-chain V region and the L-chain V region of an antibody.
  • the H-chain V region and the L-chain V region are linked via a linker, preferably via a peptide linker ( Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883 ).
  • the V regions of the H and L chains in an scFv may be derived from any of the antibodies described above.
  • Peptide linkers for linking the V regions include, for example, arbitrary single chain peptides consisting of 12 to 19 amino acid residues.
  • An scFv-encoding DNA can be obtained by using a DNA encoding an H chain or a V region and a DNA encoding an L chain or a V region of the aforementioned antibodies as templates, using PCR to amplify a DNA portion that encodes the desired amino acid sequence in the template sequence and uses primers that define the termini of the portion, and then further amplifying the amplified DNA portion with a DNA that encodes a peptide linker portion and primer pairs that link both ends of the linker to the H chain and L chain.
  • an expression vector comprising the DNA and a host transformed with the vector can be obtained according to conventional methods.
  • scFv can be obtained according to conventional methods using the host.
  • antibody fragments can be produced from the host by obtaining and expressing their genes.
  • an "antibody” encompasses such antibody fragments.
  • Antibodies bound to various molecules such as polyethylene glycol (PEG) may also be used as modified antibodies.
  • an “antibody” encompasses such modified antibodies.
  • These modified antibodies can be obtained by chemically modifying the obtained antibodies. Such methods are already established in the art.
  • Antibodies produced and expressed as above can be isolated from the inside or outside of the cells or from the hosts, and then purified to homogeneity.
  • the antibodies for use in the present disclosure can be isolated and/or purified using affinity chromatography.
  • Columns to be used for the affinity chromatography include, for example, protein A columns and protein G columns.
  • Carriers used for the protein A columns include, for example, HyperD, POROS, Sepharose FF and such.
  • other methods used for the isolation and/or purification of common proteins may be used, and are not limited in any way.
  • the antibodies used for the present invention may be isolated and/or purified by appropriately selecting and combining chromatographies in addition to affinity chromatography, filters, ultrafiltration, salting-out, dialysis, and such. Chromatographies include, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, and such. These chromatographies can be applied to high performance liquid chromatography (HPLC). Alternatively, reverse phase HPLC may be used.
  • HPLC high performance liquid chromatography
  • the measurement can be performed as follows: Specifically, 100 ⁇ l of goat anti-human IgG (TAG) diluted to 1 ⁇ g/ml with 0.1 M bicarbonate buffer (pH 9.6) is added to a 96-well plate (Nunc) and incubated overnight at 4°C to immobilize the antibody. After blocking, 100 ⁇ l of an appropriately diluted antibody of the present invention or an appropriately diluted sample comprising the antibody, and human IgG (CAPPEL) are added as a standard, and incubated for one hour at room temperature.
  • TAG goat anti-human IgG
  • CAPPEL human IgG
  • anti-IL-6 antibodies include, but are not particularly limited to, the antibodies MH166 ( Matsuda, T. et al., Eur. J. Immunol. (1998) 18, 951-956 ) and SK2 ( Sato K et al., The abstracts of the 21st Annual Meeting of the Japanese Society for Immunology (1991) 21, 166 ).
  • anti-IL-6 receptor antibodies include, but are not particularly limited to, the antibodies MR16-1 ( Tamura, T. et al., Proc. Natl. Acad. Sci. USA (1993) 90, 11.924-11928 ), PM-1 ( Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906 ), AUK12-20, AUK64-7, and AUK146-15 (International Patent Application No. WO 92-19759 ).
  • the PM-1 antibody is an example of preferred monoclonal antibodies against the human IL-6 receptor
  • the MR16-1 antibody is an example of preferred monoclonal antibodies against the mouse IL-6 receptor; however, the antibodies are not limited thereto.
  • preferred humanized anti-IL-6 receptor antibodies include a humanized PM-1 antibody (Tocilizumab; MRA).
  • Other preferred humanized anti-IL-6 receptor antibodies include, for example, the antibodies described in WO2009/041621 .
  • anti-IL-6 receptor antibodies include those that recognize the same epitope recognized by a humanized PM-1 antibody (Tocilizumab; MRA).
  • anti-gp130 antibodies include, but are not particularly limited to, the antibodies AM64 ( JP-A(Kokai) Hei 3-219894 ), 4B11, 2H4 (United States Patent Publication No. US 5571513 ), and B-P8 ( JP-A (Kokai) Hei 8-291199 ).
  • the IL-6 variants used herein are substances with the activity of binding to an IL-6 receptor and which do not transmit IL-6 biological activity. That is, the IL-6 variants compete with IL-6 to bind to IL-6 receptors, but fail to transmit IL-6 biological activity, and thus block IL-6-mediated signal transduction.
  • the IL-6 variants are produced by introducing mutation(s) by substituting amino acid residues in the amino acid sequence of IL-6.
  • the origin of IL-6 used as the base of the IL-6 variants is not limited, but is preferably human IL-6, considering antigenicity and such.
  • amino acid substitutions are performed by predicting the secondary structure of the IL-6 amino acid sequence using known molecular modeling programs (e . g ., WHATIF; Vriend et al., J. Mol. Graphics (1990) 8, 52-56 ), and further assessing the influence of the substituted amino acid residue(s) on the whole molecule.
  • known molecular modeling programs e . g ., WHATIF; Vriend et al., J. Mol. Graphics (1990) 8, 52-56 .
  • PCR methods are carried out using a nucleotide sequence encoding a human IL-6 gene as a template, and mutations are introduced to cause amino acids substitutions, and thus genes encoding IL-6 variants are obtained. If needed, this gene is inserted into an appropriate expression vector, and the IL-6 variant can be obtained by applying the aforementioned methods for expression, production, and purification of recombinant antibodies.
  • IL-6 variants are those disclosed in Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93 , Savino et al., EMBO J. (1994) 13, 1357-1367 , WO 96/18648 , and WO 96/17869 .
  • the IL-6 receptor partial peptides are peptides that comprise part or all of the amino acid sequence of the region of the IL-6 receptor amino acid sequence that is involved in the binding between the IL-6 and IL-6 receptor.
  • Such peptides usually comprise ten to 80, preferably 20 to 50, more preferably 20 to 40 amino acid residues.
  • the IL-6 receptor partial peptides can be produced according to generally known methods, for example, genetic engineering techniques or peptide synthesis methods, by specifying the region of the IL-6 receptor amino acid sequence that is involved in the binding between the IL-6 and IL-6 receptor, and using a portion or entirety of the amino acid sequence of the specified region.
  • a DNA sequence encoding the desired peptide is inserted into an expression vector, and then the peptide can be obtained by applying the aforementioned methods for expressing, producing, and purifying recombinant antibodies.
  • peptide synthesis methods for example, solid phase synthesis methods or liquid phase synthesis methods, may be used.
  • the peptides can be synthesized according to the method described in " Continuation of Development of Pharmaceuticals, Vol. 14, Peptide Synthesis (in Japanese) (ed. Haruaki Yajima, 1991, Hirokawa Shoten )".
  • the following method can be employed: the amino acid corresponding to the C terminus of the peptide to be synthesized is bound to a support that is insoluble in organic solvents, then the peptide strand is elongated by alternately repeating (1) the reaction of condensing amino acids, whose ⁇ -amino groups and branch chain functional groups are protected with appropriate protecting groups, one at a time in a C- to N-terminal direction; and (2) the reaction of removing the protecting groups from the ⁇ -amino groups of the resin-bound amino acids or peptides.
  • Solid phase peptide synthesis is broadly classified into the Boc method and the Fmoc method, depending on the type of protecting groups used.
  • the peptide strand is cleaved from its support.
  • hydrogen fluoride or trifluoromethane sulfonic acid are generally used for the Boc method
  • TFA is generally used for the Fmoc method.
  • Boc method for example, the above-mentioned protected peptide resin is treated with hydrogen fluoride in the presence of anisole. Then, the peptide is recovered by removing the protecting groups and cleaving the peptide from its support. By freeze-drying the recovered peptide, a crude peptide can be obtained.
  • the deprotection reaction and the reaction to cleave the peptide strand from the support can be performed in TFA using a method similar to those described above, for example.
  • Obtained crude peptides can be separated and/or purified by applying HPLC. Elution may be performed under optimum conditions using a water-acetonitrile solvent system, which is generally used for protein purification. The fractions corresponding to the peaks of the obtained chromatographic profile are collected and freeze-dried. Thus, purified peptide fractions are identified by molecular weight analysis via mass spectrum analysis, amino acid composition analysis, amino acid sequence analysis, or such.
  • neural invasion refers to a mode of cell invasion and growth in nerve tissues by cancer cells or other types of cells, and this sometimes accompanies tissue destruction (destructive growth) or such.
  • preferred neural invasion includes neural invasion by cancer cells.
  • the cancer may be of any type including pancreatic cancer, stomach cancer, prostatic cancer, head and neck cancer, breast cancer, lung cancer, colon cancer, and ovarian cancer.
  • pancreatic cancer cells It is possible to suppress neural invasion in either the central or peripheral direction.
  • it is preferable to suppress neural invasion in the central direction for example, neural invasion from a nerve damage site to the central side
  • pancreatic cancer cells tend to invade nerves in the central direction.
  • suppression of neural invasion refers to suppression of the occurrence of neural invasion, reduction of the incidence of neural invasion, reduction of the distance of neural invasion, retardation of the rate of neural invasion, or the like.
  • Various symptoms associated with neural invasion can be treated or suppressed by suppressing neural invasion using an IL-6 inhibitor described herein.
  • the present invention also includes agents for treating or suppressing various symptoms associated with neural invasion, which comprise an IL-6 inhibitor.
  • the therapeutic agents for pancreatic cancer described herein can be used in the treatment and/or prevention of pancreatic cancer.
  • treatment of pancreatic cancer refers to suppression of the development of pancreatic cancer, reduction of the incidence of pancreatic cancer, suppression of the growth of pancreatic cancer cells, shrinkage of pancreatic cancer tissues, amelioration of pancreatic cancer symptoms, suppression of pancreatic cancer metastasis, or the like.
  • IL-6 inhibitors used herein can be assessed, for example, using the activity of inhibiting the signaling as an indicator. However, the assessment methods are not limited thereto.
  • the activity of IL-6 inhibitors in inhibiting the signal transduction can be evaluated by conventional methods. Specifically, IL-6 is added to cultures of IL-6-dependent human myeloma cell lines (S6B45 and KPMM2), human Lennert T lymphoma cell line KT3, or IL-6-dependent cell line MH60.BSF2; and the 3 H-thymidine uptake by the IL-6-dependent cells is measured in the presence of an IL-6 inhibitor.
  • IL-6 receptor-expressing U266 cells are cultured, and 125 I-labeled IL-6 and an IL-6 inhibitor are added to the culture at the same time; and then 125 I-labeled IL-6 bound to the IL-6 receptor-expressing cells is quantified.
  • a negative control group that does not contain an IL-6 inhibitor is included in the assay system described above. The activity of the IL-6 inhibitor to inhibit IL-6 can be evaluated by comparing the results of both groups.
  • Subjects to be administered with the therapeutic or suppressing agents of the present invention are mammals.
  • the mammals are preferably humans.
  • the therapeutic or suppressing agents of the present invention can be administered as pharmaceuticals, and may be administered systemically or locally via oral or parenteral administration.
  • intravenous injections such as drip infusions, intramuscular injections, intraperitoneal injections, subcutaneous injections, suppositories, enemas, oral enteric tablets, or the like can be selected.
  • Appropriate administration methods can be selected depending on a patient's age and symptoms.
  • the effective dose per administration is selected from the range of 0.01 to 100 mg/kg body weight. Alternatively, the dose may be selected from the range of 1 to 1000 mg/patient, preferably from the range of 5 to 50 mg/patient.
  • a preferred dose and administration method are as follows: For example, when an anti-IL-6 receptor antibody is used, the effective dose is an amount such that free antibody is present in the blood. Specifically, a dose of 0.5 to 40 mg/kg body weight/month (four weeks), preferably 1 to 20 mg/kg body weight/month is administered via an intravenous injection such as a drip infusion, subcutaneous injection or such, once to several times a month, for example, twice a week, once a week, once every two weeks, or once every four weeks.
  • the administration schedule may be adjusted by, for example, extending the administration interval of twice a week or once a week to once every two weeks, once every three weeks, or once every four weeks, while monitoring the condition of the patient and changes in the blood test values.
  • the therapeutic agents and suppressing agents of the present invention may contain pharmaceutically acceptable carriers such as preservatives and stabilizers.
  • pharmaceutically acceptable carrier refers to a material that can be administered in combination with the above agents.
  • Such pharmaceutically acceptable materials include, for example, sterile water, physiological saline, stabilizers, excipients, buffers, preservatives, detergents, chelating agents (EDTA and such), and binders.
  • detergents include non-ionic detergents, and typical examples of such include sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate, and sorbitan monopalmitate; glycerin fatty acid esters such as glycerin monocaprylate, glycerin monomyristate and glycerin monostearate; polyglycerin fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, and decaglyceryl monolinoleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and polyoxyethylene sorbitan tristearate; polyoxyethylene sorbit fatty acid esters such as polyoxyethylene sorbitan fatty acid est
  • Detergents also include anionic detergents, and typical examples of such include, for example, alkylsulfates having an alkyl group with ten to 18 carbon atoms, such as sodium cetylsulfate, sodium lautylsulfate, and sodium oleylsulfate; polyoxyethylene alkyl ether sulfates in which the alkyl group has ten to 18 carbon atoms and the average molar number of added ethylene oxide is 2 to 4, such as sodium polyoxyethylene lauryl sulfate; alkyl sulfosuccinate ester salts having an alkyl group with eight to 18 carbon atoms, such as sodium lauryl sulfosuccinate ester; natural detergents, for example, lecithin; glycerophospholipids; sphingo-phospholipids such as sphingomyelin; and sucrose fatty acid esters in which the fatty acids have 12 to 18 carbon atoms.
  • detergents described above can be combined and added to the agents described herein.
  • Detergents that are preferably used in the preparations of the present invention include polyoxyethylene sorbitan fatty acid esters, such as polysorbates 20, 40, 60, and 80. Polysorbates 20 and 80 are particularly preferred. Polyoxyethylene polyoxypropylene glycols, such as poloxamer (Pluronic F-68 ® and such), are also preferred.
  • the amount of detergent added varies depending on the type of detergent used.
  • the amount is in general in the range of 0.001 to 100 mg/ml, preferably in the range of 0.003 to 50 mg/ml, more preferably in the range of 0.005 to 2 mg/ml.
  • buffers include phosphate, citrate buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, capric acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid, and other organic acids; and carbonic acid buffer, Tris buffer, histidine buffer, and imidazole buffer.
  • Liquid preparations may be formulated by dissolving the agents in aqueous buffers known in the field of liquid preparations.
  • the buffer concentration is in general in the range of 1 to 500 mM, preferably in the range of 5 to 100 mM, more preferably in the range of 10 to 20 mM.
  • the agents described herein may also comprise other low-molecular-weight polypeptides; proteins such as serum albumin, gelatin, and immunoglobulin; amino acids; sugars and carbohydrates such as polysaccharides and monosaccharides, sugar alcohols, and such.
  • amino acids include basic amino acids, for example, arginine, lysine, histidine, and ornithine, and inorganic salts of these amino acids (preferably hydrochloride salts, and phosphate salts, namely phosphate amino acids).
  • inorganic salts preferably hydrochloride salts, and phosphate salts, namely phosphate amino acids.
  • the pH is adjusted to a preferred value by adding appropriate physiologically acceptable buffering substances, for example, inorganic acids, and in particular hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, and formic acid, and salts thereof.
  • phosphate is particularly beneficial because it gives quite stable freeze-dried products.
  • Phosphate is particularly advantageous when preparations do not substantially contain organic acids, such as malic acid, tartaric acid, citric acid, succinic acid, and fumaric acid, or do not contain corresponding anions (malate ion, tartrate ion, citrate ion, succinate ion, fumarate ion, and such).
  • Preferred amino acids are arginine, lysine, histidine, and ornithine.
  • Acidic amino acids can also be used, for example, glutamic acid and aspartic acid, and salts thereof (preferably sodium salts); neutral amino acids, for example, isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine, and alanine; and aromatic amino acids, for example, phenylalanine, tyrosine, tryptophan, and its derivative, N-acetyl tryptophan.
  • neutral amino acids for example, isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine, and alanine
  • aromatic amino acids for example, phenylalanine, tyrosine, tryptophan, and its derivative, N-acetyl tryptophan.
  • sugars and carbohydrates such as polysaccharides and monosaccharides include, for example, dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose.
  • sugar alcohols include, for example, mannitol, sorbitol, and inositol.
  • the agents of the present invention are prepared as aqueous solutions for injection
  • the agents may be mixed with, for example, physiological saline, and/or isotonic solution containing glucose or other auxiliary agents (such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride).
  • glucose or other auxiliary agents such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride.
  • the aqueous solutions may be used in combination with appropriate solubilizing agents such as alcohols (ethanol and such), polyalcohols (propylene glycol, PEG, and such), or non-ionic detergents (polysorbate 80 and HCO-50).
  • the agents may further comprise, if required, diluents, solubilizers, pH adjusters, soothing agents, sulfur-containing reducing agents, antioxidants, and such
  • the sulfur-containing reducing agents include, for example, compounds comprising sulfhydryl groups, such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having one to seven carbon atoms.
  • sulfhydryl groups such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having one to seven carbon atoms.
  • the antioxidants as described herein include, for example, erythorbic acid, dibutylhydroxy toluene, butylhydroxy anisole, ⁇ -tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as disodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.
  • EDTA disodium ethylenediamine tetraacetate
  • the agents may be encapsulated in microcapsules (microcapsules of hydroxymethylcellulose, gelatin, poly[methylmethacrylic acid] or such) or prepared as colloidal drug delivery systems (liposome, albumin microspheres, microemulsion, nano-particles, nano-capsules, and such) (see “ Remington's Pharmaceutical Science 16th edition", Oslo Ed., 1980, and the like ).
  • methods for preparing agents as sustained-release agents are also known, and are applicable to the present invention ( Langer et al., J. Biomed. Mater. Res. 1981, 15: 167-277 ; Langer, Chem. Tech. 1982, 12: 98-105 ; U.S. Patent No. 3,773,919 ; European Patent Application No. ( EP) 58,481 ; Sidman et al., Biopolymers 1983, 22: 547-556 ; and EP 133,988 ).
  • Pharmaceutically acceptable carriers used are appropriately selected from those described above or combined depending on the type of dosage form, but are not limited thereto.
  • the present disclosure relates to methods for suppressing neural invasion in a subject who has developed or can develop neural invasion, which comprise the step of administering an IL-6 inhibitor to the subject.
  • the present disclosure also relates to methods for treating and/or preventing pancreatic cancer in a subject who has developed or can develop pancreatic cancer, which comprise the step of administering an IL-6 inhibitor to the subject.
  • the "subject” refers to the organisms or organism body parts to be administered with a therapeutic or suppressing agent of the present invention.
  • the organisms include animals (for example, human, domestic animal species, and wild animals) but are not particularly limited.
  • the "organism body parts” preferably include disease sites, but are not particularly limited thereto.
  • oral administration includes, for example, administration of oral agents.
  • oral agents include, for example, granules, powders, tablets, capsules, solutions, emulsions, and suspensions.
  • Parenteral administration includes, for example, administration of injections.
  • injections include, for example, subcutaneous injections, intramuscular injections, and intraperitoneal injection.
  • the effects of the methods described herein can be achieved by introducing genes comprising oligonucleotides to be administered to living bodies using gene therapy techniques.
  • the agents described herein may be administered locally to intended areas of treatment.
  • the agents can be administered by local injection during surgery, use of catheters, or targeted gene delivery of DNAs encoding peptides described herein.
  • an agent described herein may be administered as a portion of a pharmaceutical composition, together with at least one other agent (for example, other neural invasion-suppressing agents and other therapeutic agents for pancreatic cancer):
  • at least one other agent for example, other neural invasion-suppressing agents and other therapeutic agents for pancreatic cancer:
  • the agents described herein and other agents may be administered substantially simultaneously.
  • Capan-1 and BxPC-3 which are human pancreatic cancer cell lines, were purchased from the American Type Culture Collection (ATCC). According to the manual recommended by ATCC, the cells were cultured and passaged using an incubator capable of maintaining a temperature of 37°C under 5% CO 2 .
  • Cells were harvested from dishes, and viable cells were counted using trypan blue and a hemocytometer.
  • hrIL6 Human recombinant IL-6
  • R&D systems hrIL6
  • serum-free culture medium 600 ⁇ l of the hrIL6 vehicles was added to the lower chambers.
  • 100 ⁇ l of a cell suspension (2 x 10 6 cells/ml) was added to the upper chambers. After 24 hours of incubation, cells that passed through the pores were counted. This assay was carried out twelve times for each group. The cell count was divided by the average number of cells that passed through the pores at 0 ng/ml hrIL6. The resulting normalized values were recorded.
  • Western blotting was carried out using the following primary antibodies: anti-phosphorylated STAT3 antibody (Santa Cruz), anti-STAT3 antibody (Santa Cruz), anti-phosphorylated Erk1/2 antibody (Cell Signaling), anti-Erkl/2 antibody (Cell Signaling), anti-phosphorylated Akt antibody (Cell Signaling), anti-Akt antibody (Cell Signaling), and anti-actin antibody (Santa Cruz). Fluorescent immunostaining was carried out using the following primary antibodies: anti-S100 antibody (DAKO) and anti-mouse IL-6 antibody (Santa Cruz). Nuclear staining was performed using DRAQ5 (AXXORA). Immunostaining was performer using an anti-phosphorylated STAT3 antibody (Santa Cruz).
  • Cell lysates were prepared using a lysate buffer (20 mM Hepes-NaOH (pH 7.0), 0.5% NP-40, 15% Glycerol, 300 mM NaCl, 1 mM EDTA, 10 mM NaF). The protein concentration was determined using a BCA Protein Assay Kit (PIERCE). Then, cell lysates containing 20 mg of protein were electrophoresed in 7.5% or 12% acrylamide gels. The proteins were transferred onto a polyvinylidene difluoride membrane (Millipore). An antibody was added to the membrane, and the protein expression was visualized using Enhanced Chemiluminescence Reagent (Amersham Biosciences).
  • Antigen activation was performed by heat treatment at 95°C for ten minutes in 10 mM citrate buffer using a microwave. DAB was used for the chromogenic reaction.
  • 26 sciatic nerves were isolated from 26 mice, and used as samples.
  • the numbers of cancer cells and phosphorylated STAT3-positive cancer cells were counted in each visual field using a 40x objective lens.
  • the labeling index was determined according to the following formula: Number of phosphorylated STAT ⁇ 3 - positive cancer cells / Number of cancer cells .
  • mice with severe combined immune deficiency were used.
  • the mice were anesthetized by administering barbital at 50 mg/kg into their peritoneal cavities.
  • 2.5 ⁇ l of a suspension of cancer cells 1.0 x 10 4 cells/ ⁇ l
  • the sciatic nerves into which cancer cells were injected were isolated.
  • tissue samples were prepared, the nerves were allowed to stand in 4% paraformaldehyde at 4°C for one whole day and night to fix them.
  • the fixed sciatic nerves were sliced into 3- ⁇ m sections, and then immunostained or hematoxylin/eosin-stained to measure the distance of neural invasion. Using an objective micrometer (Sankei Co.), the longitudinal length of the whole tumor was measured in the longitudinally sliced thin sections of the nerves to determine the distance of neural invasion. Tissue mRNA was extracted from tissue samples prepared by crushing with a Multi-Beads Shocker (Yasui Kikai Co.) immediately after harvesting the tissues.
  • IL6R human IL-6 ⁇ receptor
  • gp130 human IL-6 ⁇ receptor
  • GAPDH human GAPDH
  • mouse IL-6 mouse EGF
  • mouse GAPDH mouse GAPDH
  • siRNAs produced by Ambion The following siRNAs were used: human IL6R siRNA, human gp130 siRNA, and Negative Control #1 siRNA.
  • 2 x 10 5 cancer cells were plated in 3.5-cm dishes. After 48 hours of incubation, 20 ⁇ M of siRNA and 8 ⁇ l of DharmaFECT transfection reagent 4 (Dharmacon) were added to the cells. After 24 hours, the cells were harvested and used for mRNA expression analysis or for preparation of the neural invasion model.
  • DharmaFECT transfection reagent 4 Dharmacon
  • the analysis software used was STATVIEW 5.0.
  • the difference between average values was evaluated by two-sided Student-t test. Error bars in the figures indicate the standard deviation.
  • IL-6 ⁇ receptor IL-6 ⁇ receptor
  • gp130 IL-6 ⁇ receptor
  • IL-6 Using human recombinant IL-6, the effects of IL-6 on growth activity, chemotactic activity, and migratory activity of the human pancreatic cancer cell lines were assessed by monitoring the cell count over time ( Figs. 2A and 2B ), by chemotaxis assay ( Fig. 2C ), and by wound healing assay ( Fig. 2D ). It was demonstrated that IL-6 enhances the chemotactic and migratory activities of the pancreatic cancer cell lines, while IL-6 has no effect on the cell growth.
  • IL-6 human recombinant IL-6
  • rhIL6 human recombinant IL-6
  • IL-6 the effect of IL-6 on intracellular signaling of the human pancreatic cancer Capan-1 cell line was assessed by Western blotting for phosphorylated STAT3 (pSTAT3) ( Fig. 3A ), phosphorylated Erk1/2 (pErk1/2) ( Fig. 3B ), and phosphorylated Akt (pAkt) ( Fig. 3C ).
  • the intracellular expression levels of phosphorylated STAT3 protein and phosphorylated Erk1/2 proteins were clearly elevated 15 minutes and one hour after addition of rhIL6, respectively.
  • IL-6 had no effect on the expression of phosphorylated Akt.
  • the distance of neural invasion is an important factor in the mode of invasion of pancreatic cancer. It is essential to create a mouse model for neural invasion that reproduces neural invasion and allows measuring the distance of neural invasion in order to assess therapeutic methods for controlling this vital mode of tumor invasion in pancreatic cancer.
  • a neural invasion model was prepared by directly introducing cells of the human pancreatic cancer Capan-1 cell line into the sciatic nerves of immunodeficient mice. Macroscopically, the nerves had a rough surface at the site of neural invasion, and they were clearly thicker than the normal nerve ( Fig. 4A ). Histologically, the distance of neural invasion one week after the injection was clearly longer than the distance of intraneural diffusion of injected Capan-1, and the distance was increased over time ( Fig. 4B ). The neural invasion expanded from the injection site toward the central side ( Fig. 4C ). These characteristics are similar to those of the neural invasion of human pancreatic cancer.
  • mIL6 mouse IL-6
  • IL6-positive granules were found to be present in the same area in which cells are positive for S100, which is a marker of Schwann cells ( Fig. 5C ).
  • S100 which is a marker of Schwann cells
  • mIL6-secreting cells include Schwann cells.
  • EGF is known to be expressed at a high level upon nerve damage.
  • mEGF mouse EGF
  • phosphorylated STAT3 (pSTAT3) protein, which is important for the intracellular signaling of IL-6, in pancreatic cancer cells was assessed by immunostaining.
  • the expression of phosphorylated STAT3 was found to be enhanced on the central side of the neural invasion site ( Fig. 6 ). This is consistent with the result that the expression of IL-6 is enhanced in nerve tissues on the central side of the neural invasion site.
  • gp130 is essential for the intracellular signaling of IL-6.
  • siRNA siRNA
  • gp130 mRNA expression was knocked down in a pancreatic cancer cell line.
  • the distance of neural invasion was decreased ( Fig. 7 ). This result shows that signals mediated by gp130 including those derived from IL-6 are essential for neural invasion.
  • IL-6 receptor IL-6 receptor
  • siRNA siRNA
  • IL6R mRNA expression was knocked down in a pancreatic cancer cell line.
  • the neural invasion model was prepared using the knockdown cells, the distance of neural invasion was decreased ( Fig. 8 ). This result shows that signals mediated by IL-6 are essential for neural invasion.
  • JAK inhibitor or an anti-IL-6 receptor antibody was administered to neural invasion model mice to assess the effect of these inhibitors on neural invasion.
  • the JAK inhibitor AG490 (CALBIOCHEM), which inhibits STAT3 phosphorylation, was dissolved in DMSO, and then diluted with physiological saline to prepare an AG490 solution in 1% DMSO. From two days after preparation of the neural invasion model, 0.5 mg of AG490 was administered into the peritoneal cavities of the mice every day. Two weeks after the model preparation, the sciatic nerves into which cancer cells were injected were isolated to determine the distance of neural invasion. For the control group, 1% DMSO was administered in the same manner as described above. The number of mice used was seven in each of the AG490 and DMSO groups.
  • an anti-IL-6 receptor antibody (tocilizumab; Chugai Pharmaceutical Co.), which inhibits the human IL-6 receptor, was dissolved in physiological saline.
  • the anti-IL-6 inhibitory antibody was administered to the mice at 5 ⁇ g/g twice a week.
  • the sciatic nerves into which cancer cells were injected were isolated to determine the distance of neural invasion.
  • human IgG Sigma
  • dissolved at 5 ⁇ g/g in physiological saline was administered in the same manner as described above. The number of mice used was six in the anti-IL-6 receptor antibody group and four in the control group.
  • the analysis software used was STATVIEW 5.0.
  • the difference between average values was evaluated by two-sided Student-t test. Error bars in the figures indicate the standard deviation.
  • an anti-human IL-6 receptor antibody was administered twice a week for two weeks to inhibit the action of human IL-6. This resulted in suppression of neural invasion ( Fig. 9B ). This finding indicates that neural invasion by human pancreatic cancer was inhibited by the anti-human IL-6 receptor antibody.
  • pancreatic cancer can be suppressed by administering an anti-IL-6 receptor antibody. Furthermore, it was also shown that pancreatic cancer can be treated by administering an anti-IL-6 receptor antibody.

Claims (9)

  1. Agent pour une utilisation dans la suppression de l'invasion neurale par une cellule de cancer pancréatique, qui comprend en tant que principe actif un anticorps anti-récepteur de l'IL-6 qui bloque la liaison entre l'IL-6 et un récepteur de l'IL-6.
  2. Utilisation d'un anticorps anti-récepteur de l'IL-6 qui bloque la liaison entre l'IL-6 et un récepteur de l'IL-6 dans la production d'un agent destiné à la suppression de l'invasion neurale par une cellule de cancer pancréatique.
  3. Anticorps anti-récepteur de l'IL-6 qui bloque la liaison entre l'IL-6 et un récepteur de l'IL-6 pour une utilisation dans la suppression de l'invasion neurale par une cellule de cancer pancréatique.
  4. Agent pour une utilisation selon la revendication 1, où un anticorps anti-récepteur de l'IL-6 supprime l'invasion neurale dans la direction centrale.
  5. Utilisation selon la revendication 2, où l'anticorps anti-récepteur de l'IL-6 supprime l'invasion neurale dans la direction centrale.
  6. Anticorps anti-récepteur de l'IL-6 pour une utilisation selon la revendication 3, où l'anticorps anti-récepteur de l'IL-6 supprime l'invasion neurale dans la direction centrale.
  7. Agent pour une utilisation selon la revendication 1 ou 4, où l'anticorps anti-récepteur de l'IL-6 est un anticorps chimérique, humanisé, ou humain.
  8. Utilisation selon la revendication 2 ou 5, où l'anticorps anti-récepteur de l'IL-6 est un anticorps chimérique, humanisé, ou humain.
  9. Anticorps anti-récepteur de l'IL-6 pour une utilisation selon la revendication 3 ou 6, où l'anticorps anti-récepteur de l'IL-6 est un anticorps chimérique, humanisé, ou humain.
EP09758415.5A 2008-06-05 2009-06-05 Inhibiteur de neuro-invasion Active EP2305306B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2008147944 2008-06-05
PCT/JP2009/060314 WO2009148148A1 (fr) 2008-06-05 2009-06-05 Inhibiteur de neuro-invasion

Publications (3)

Publication Number Publication Date
EP2305306A1 EP2305306A1 (fr) 2011-04-06
EP2305306A4 EP2305306A4 (fr) 2012-08-29
EP2305306B1 true EP2305306B1 (fr) 2016-02-10

Family

ID=41398219

Family Applications (1)

Application Number Title Priority Date Filing Date
EP09758415.5A Active EP2305306B1 (fr) 2008-06-05 2009-06-05 Inhibiteur de neuro-invasion

Country Status (9)

Country Link
US (1) US10717781B2 (fr)
EP (1) EP2305306B1 (fr)
JP (1) JP5544290B2 (fr)
KR (1) KR101665729B1 (fr)
CN (2) CN102256623A (fr)
CA (1) CA2728243C (fr)
HK (1) HK1214514A1 (fr)
TW (2) TWI528973B (fr)
WO (1) WO2009148148A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11851486B2 (en) 2017-05-02 2023-12-26 National Center Of Neurology And Psychiatry Method for predicting and evaluating therapeutic effect in diseases related to IL-6 and neutrophils

Families Citing this family (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2322216A1 (fr) 1997-03-21 2011-05-18 Chugai Seiyaku Kabushiki Kaisha Agent thérapeutique ou préventif pour les maladies liées aux lymphocytes T sensibilisés comprenant l'antagoniste IL-6 en tant qu'ingrédient actif
UA80091C2 (en) 2001-04-02 2007-08-27 Chugai Pharmaceutical Co Ltd Remedies for infant chronic arthritis-relating diseases and still's disease which contain an interleukin-6 (il-6) antagonist
CN1638798A (zh) * 2002-02-14 2005-07-13 中外制药株式会社 包含抗体的溶液制剂
GB2401040A (en) * 2003-04-28 2004-11-03 Chugai Pharmaceutical Co Ltd Method for treating interleukin-6 related diseases
JPWO2005090405A1 (ja) 2004-03-24 2008-04-17 中外製薬株式会社 インターロイキン−６受容体に対するヒト型化抗体のサブタイプ
WO2007043641A1 (fr) * 2005-10-14 2007-04-19 Fukuoka University Inhibiteur de dysfonctionnement d'îlots transplantés dans un transplant d'îlots
AU2006305119B2 (en) * 2005-10-21 2012-12-20 Chugai Seiyaku Kabushiki Kaisha Agents for treating cardiopathy
AR057582A1 (es) * 2005-11-15 2007-12-05 Nat Hospital Organization Agentes para suprimir la induccion de linfocitos t citotoxicos
WO2007061029A1 (fr) * 2005-11-25 2007-05-31 Keio University Agent thérapeutique pour le cancer de la prostate
CN101370521A (zh) * 2006-01-27 2009-02-18 学校法人庆应义塾 伴有脉络膜血管生成的疾病的治疗药
EP4342995A2 (fr) 2006-03-31 2024-03-27 Chugai Seiyaku Kabushiki Kaisha Procédés de régulation de la pharmacocinétique sanguine d'anticorps
CA2648644C (fr) * 2006-04-07 2016-01-05 Osaka University Promoteur de regeneration musculaire
CN101646459B (zh) 2007-01-23 2014-02-12 国立大学法人信州大学 慢性排斥反应抑制剂
MX2010003450A (es) 2007-09-26 2010-04-27 Chugai Pharmaceutical Co Ltd Region constante de anticuerpo modificada.
EP3127921A1 (fr) 2007-09-26 2017-02-08 Chugai Seiyaku Kabushiki Kaisha Procédé de modification d'un anticorps par point isoélectrique par substitution d'acide aminé dans cdr
PE20091174A1 (es) 2007-12-27 2009-08-03 Chugai Pharmaceutical Co Ltd Formulacion liquida con contenido de alta concentracion de anticuerpo
KR102269708B1 (ko) 2008-04-11 2021-06-25 추가이 세이야쿠 가부시키가이샤 복수 분자의 항원에 반복 결합하는 항원 결합 분자
TWI528973B (zh) 2008-06-05 2016-04-11 Chugai Pharmaceutical Co Ltd Nerve infiltration inhibitor
TWI440469B (zh) 2008-09-26 2014-06-11 Chugai Pharmaceutical Co Ltd Improved antibody molecules
KR101436219B1 (ko) * 2009-10-26 2014-09-01 에프. 호프만-라 로슈 아게 글리코실화된 면역글로불린의 제조 방법
WO2011149046A1 (fr) 2010-05-28 2011-12-01 独立行政法人国立がん研究センター Agent thérapeutique contre le cancer du pancréas
RS63800B1 (sr) * 2010-05-28 2022-12-30 Chugai Pharmaceutical Co Ltd Poboljšanje antitumorskog odgovora t ćelije
BR112013011305A2 (pt) 2010-11-08 2017-07-25 Chugai Pharmaceutical Co Ltd método de tratamento de uma disfunção mediada por il-6, método de tratamento de artrite reumatóide, artigo de fabricação, método de inibição da progressão de dano estrutural articular, composição farmacêutica, método de tratamento de artrite idiopática juvenil (jia) e método de tratamento de arterite de células gigantes (gca)
AU2011337704B2 (en) 2010-11-30 2017-06-15 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule capable of binding to plurality of antigen molecules repeatedly
WO2014200018A1 (fr) 2013-06-11 2014-12-18 独立行政法人 国立精神・神経医療研究センター Procédé permettant de prédire le pronostic post-thérapie d'un patient atteint de sclérose en plaques à périodes progressives et rémittentes (spppr) et procédé permettant de déterminer l'applicabilité d'une nouvelle thérapie
KR102248581B1 (ko) 2013-07-04 2021-05-06 에프. 호프만-라 로슈 아게 혈청 샘플 중 항-약물 항체를 검출하기 위한 간섭-억제된 면역분석
CA2972393A1 (fr) 2015-02-27 2016-09-01 Chugai Seiyaku Kabushiki Kaisha Composition pour le traitement de maladies associees a il-6
ES2888801T3 (es) 2015-05-19 2022-01-07 Nat Center Neurology & Psychiatry Método para determinar la aplicación de una terapia nueva en pacientes con esclerosis múltiple (EM)
EP3305325A4 (fr) 2015-06-04 2019-01-16 National Center of Neurology and Psychiatry Agent thérapeutique contre les maladies mentales ayant un inhibiteur d'il-6 comme principe actif
US11484591B2 (en) 2016-02-22 2022-11-01 Ohio State Innovation Foundation Chemoprevention using controlled-release formulations of anti-interleukin 6 agents, synthetic vitamin A analogues or metabolites, and estradiol metabolites
AU2018234844B2 (en) 2017-03-17 2024-01-25 Ohio State Innovation Foundation Nanoparticles for delivery of chemopreventive agents
EP3698808A4 (fr) 2017-10-20 2021-07-14 Hyogo College Of Medicine Composition médicinale contenant un anticorps du recepteur anti-il -6 pour prévenir l'adhérence post-chirurgicale
WO2020201362A2 (fr) 2019-04-02 2020-10-08 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes de prédiction et de prévention du cancer chez des patients ayant des lésions prémalignes
CN113796349A (zh) * 2021-08-06 2021-12-17 南通大学 一种乳腺癌侵袭神经及诱发疼痛的动物模型及其制备方法

Family Cites Families (121)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
IE52535B1 (en) 1981-02-16 1987-12-09 Ici Plc Continuous release pharmaceutical compositions
JPS58201994A (ja) 1982-05-21 1983-11-25 Hideaki Hagiwara 抗原特異的ヒト免疫グロブリンの生産方法
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
HUT35524A (en) 1983-08-02 1985-07-29 Hoechst Ag Process for preparing pharmaceutical compositions containing regulatory /regulative/ peptides providing for the retarded release of the active substance
GB8607679D0 (en) 1986-03-27 1986-04-30 Winter G P Recombinant dna product
US5171840A (en) 1988-01-22 1992-12-15 Tadamitsu Kishimoto Receptor protein for human B cell stimulatory factor-2
US5670373A (en) * 1988-01-22 1997-09-23 Kishimoto; Tadamitsu Antibody to human interleukin-6 receptor
US5530101A (en) * 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5216128A (en) * 1989-06-01 1993-06-01 Yeda Research And Development Co., Ltd. IFN-β2/IL-6 receptor its preparation and pharmaceutical compositions containing it
JP2998976B2 (ja) 1989-07-20 2000-01-17 忠三 岸本 ヒトインタ―ロイキン―６レセプターに対する抗体
JP2898064B2 (ja) 1989-08-03 1999-05-31 忠三 岸本 ヒトｇｐ１３０蛋白質
JPH03155795A (ja) 1989-11-13 1991-07-03 Chuzo Kishimoto マウス・インターロイキン―６レセプター蛋白質
JP2898040B2 (ja) 1990-01-26 1999-05-31 忠三 岸本 ｇｐ１３０蛋白質に対する抗体
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
ATE185601T1 (de) 1990-07-10 1999-10-15 Cambridge Antibody Tech Verfahren zur herstellung von spezifischen bindungspaargliedern
DK0814159T3 (da) 1990-08-29 2005-10-24 Genpharm Int Transgene, ikke-humane dyr, der er i stand til at danne heterologe antistoffer
DE122009000019I1 (de) * 1991-04-25 2009-07-16 Chugai Seiyaku K K 5 1 Rekombinierte humane antikörper gegen den humanen interleukin-6 rezeptor
DK0605522T3 (da) 1991-09-23 2000-01-17 Medical Res Council Fremgangsmåde til fremstilling af humaniserede antistoffer
EP0661985B1 (fr) 1991-11-08 1999-07-07 The Board Of Trustees Of The Leland Stanford Junior University Peptides capables de se lier aux molecules cd8 sur les precurseurs de ctl
ATE463573T1 (de) 1991-12-02 2010-04-15 Medimmune Ltd Herstellung von autoantikörpern auf phagenoberflächen ausgehend von antikörpersegmentbibliotheken
JPH07503132A (ja) 1991-12-17 1995-04-06 ジェンファーム　インターナショナル，インコーポレイティド 異種抗体を産生することができるトランスジェニック非ヒト動物
CA2131151A1 (fr) 1992-03-24 1994-09-30 Kevin S. Johnson Methodes de production d'elements formant des paires specifiques de liaison
ES2301158T3 (es) 1992-07-24 2008-06-16 Amgen Fremont Inc. Produccion de anticuerpos xenogenicos.
FR2694767B1 (fr) 1992-08-13 1994-10-21 Innotherapie Lab Sa Anticorps monoclonaux anti-IL6R, et leurs applications.
US5648267A (en) 1992-11-13 1997-07-15 Idec Pharmaceuticals Corporation Impaired dominant selectable marker sequence and intronic insertion strategies for enhancement of expression of gene product and expression vector systems comprising same
JP3525221B2 (ja) * 1993-02-17 2004-05-10 味の素株式会社 免疫抑制剤
US5468772A (en) 1993-03-10 1995-11-21 Pharmagenesis, Inc. Tripterinin compound and method
EP0754225A4 (fr) 1993-04-26 2001-01-31 Genpharm Int Animaux transgeniques capables de produire des anticorps heterologues
TW458985B (en) * 1993-05-31 2001-10-11 Chugai Pharmaceutical Co Ltd Reconstructed human antibody against human interleukin-6
JP3614183B2 (ja) 1993-05-31 2005-01-26 中外製薬株式会社 ヒトインターロイキン−６に対する再構成ヒト抗体
GB9313509D0 (en) 1993-06-30 1993-08-11 Medical Res Council Chemisynthetic libraries
US5888510A (en) * 1993-07-21 1999-03-30 Chugai Seiyaku Kabushiki Kaisha Chronic rheumatoid arthritis therapy containing IL-6 antagonist as effective component
US6074643A (en) * 1993-09-09 2000-06-13 Cli Oncology, Inc. Site-directed chemotherapy of metastases
AU7967294A (en) 1993-10-06 1995-05-01 Board Of Regents, The University Of Texas System A monoclonal anti-human il-6 receptor antibody
WO1995015388A1 (fr) 1993-12-03 1995-06-08 Medical Research Council Proteines et peptides de liaison recombines
WO1995033483A1 (fr) 1994-06-07 1995-12-14 Toray Industries, Inc. Agent preventif et curatif contre les maladies provoquees par la formation de fibrinoides ou de thrombus dans les poumons et modele animal pour ces maladies
US8017121B2 (en) * 1994-06-30 2011-09-13 Chugai Seiyaku Kabushika Kaisha Chronic rheumatoid arthritis therapy containing IL-6 antagonist as effective component
RU2147443C1 (ru) 1994-10-07 2000-04-20 Чугаи Сейяку Кабусики Кайся Лечебные средства против хронического ревматоидного артрита, содержащие антагонист il-6 в качестве эффективного компонента
EP1884524A3 (fr) 1994-10-21 2008-06-25 Chugai Seiyaku Kabushiki Kaisha N.a.
IT1274350B (it) 1994-12-06 1997-07-17 Angeletti P Ist Richerche Bio Antagonisti di interleuchina-6(il-6) che consistono di forme solubili del ricettore alfa di il-6, mutate nell'interfaccia che si lega a gp 130
IT1274782B (it) 1994-12-14 1997-07-24 Angeletti P Ist Richerche Bio Metodo per selezionare superagonisti, antagonisti e superantagonisti di ormoni del cui complesso recettoriale fa parte gp 130
DE69636278T2 (de) * 1995-02-13 2007-05-03 Chugai Seiyaku K.K. Inhibitor des muskelproteinabbaus enthaltend einen il-6 rezeptor antikörper
US6060293A (en) 1995-03-31 2000-05-09 Prokyon Aps Resonance driven changes in chain molecule structure
FR2733250B1 (fr) 1995-04-21 1997-07-04 Diaclone Anticorps monoclonaux anti-gp130, et leurs utilisations
EP0822830B1 (fr) 1995-04-27 2008-04-02 Amgen Fremont Inc. Anticorps anti-IL-8 dérivés de xenosouris immunisées
AU2466895A (en) 1995-04-28 1996-11-18 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5571513A (en) 1995-05-31 1996-11-05 The Board Of Regents Of The University Of Oklahoma Anti-gp130 monoclonal antibodies
RU2127117C1 (ru) 1996-06-26 1999-03-10 Филиппова Ольга Всеволодовна Способ лечения ишемической болезни сердца
GB9702944D0 (en) 1997-02-13 1997-04-02 Univ Manchester Reducing fibrosis
EP2322216A1 (fr) 1997-03-21 2011-05-18 Chugai Seiyaku Kabushiki Kaisha Agent thérapeutique ou préventif pour les maladies liées aux lymphocytes T sensibilisés comprenant l'antagoniste IL-6 en tant qu'ingrédient actif
EP0980259A1 (fr) * 1997-05-17 2000-02-23 Biogen, Inc. Utilisation d'un interrupteur de fixation cd40:cd154 pour prevenir les reponses immunitaires indesirables, en particulier le rejet de greffe
US20020187150A1 (en) * 1997-08-15 2002-12-12 Chugai Seiyaku Kabushiki Kaisha Preventive and/or therapeutic agent for systemic lupus erythematosus comprising anti-IL-6 receptor antibody as an active ingredient
JPH11180873A (ja) 1997-12-22 1999-07-06 Kaken Shoyaku Kk ＮＦ−κＢ活性阻害剤
HU225539B1 (en) 1998-03-17 2007-02-28 Chugai Pharmaceutical Co Ltd Preventives or remedies for inflammatory intestinal diseases containing as the active ingredient antibody against il-6 receptor
JP4698652B2 (ja) * 1998-03-17 2011-06-08 中外製薬株式会社 Ｉｌ−６アンタゴニストを有効成分として含有する炎症性腸疾患の予防又は治療剤
EP0972780A1 (fr) 1998-05-18 2000-01-19 Applied Research Systems ARS Holding N.V. Peptides antagonistes de l'Il-6
DK1108435T3 (da) 1998-08-24 2007-02-05 Chugai Pharmaceutical Co Ltd Forebyggende midler eller midler mod pancreatitis indeholdende anti-IL-6-receptor-antistoffer som den aktive bestanddel
JP4799516B2 (ja) * 1998-08-24 2011-10-26 中外製薬株式会社 Ｉｌ−６アンタゴニストを有効成分として含有する膵炎の予防又は治療剤
US20030130212A1 (en) 1999-01-14 2003-07-10 Rossignol Daniel P. Administration of an anti-endotoxin drug by intravenous infusion
ES2250154T3 (es) 1999-07-16 2006-04-16 Kissei Pharmaceutical Co., Ltd. Agentes que inhiben las reacciones de rechazo cronico despues de un transplante de organo.
AU3162001A (en) 1999-12-21 2001-07-03 Id-Pharma Gmbh Medicament, a method for its production and the use thereof
US6413663B1 (en) 2000-06-29 2002-07-02 Graftech Inc. Fluid permeable flexible graphite fuel cell electrode
ES2298273T3 (es) * 2000-10-25 2008-05-16 Chugai Seiyaku Kabushiki Kaisha Agentes preventivos o terapeuticos contra la psoriasis que contienen un antagonista de il-6 como su ingrediente activo.
WO2002036165A1 (fr) * 2000-10-27 2002-05-10 Chugai Seiyaku Kabushiki Kaisha Agent à base d'antagoniste d'il-6, faisant baisser le niveau des mmp-3 dans le sang
UA80091C2 (en) 2001-04-02 2007-08-27 Chugai Pharmaceutical Co Ltd Remedies for infant chronic arthritis-relating diseases and still's disease which contain an interleukin-6 (il-6) antagonist
US20050182007A1 (en) * 2001-05-18 2005-08-18 Sirna Therapeutics, Inc. RNA interference mediated inhibition of interleukin and interleukin receptor gene expression using short interfering nucleic acid (SINA)
EP2308888B1 (fr) 2001-11-14 2017-03-01 Janssen Biotech, Inc. Anticorps anti-IL-6, compositions, méthodes et utilisations associées
FR2833011B1 (fr) 2001-12-04 2004-10-29 Univ Claude Bernard Lyon Nouvelle proteine a activite inhibitrice de l'il-6
JP3856734B2 (ja) 2002-06-28 2006-12-13 株式会社日立製作所 多発性硬化症に対するインターフェロン・ベータ薬物治療の有効性予測方法
DE10231655A1 (de) 2002-07-12 2004-02-26 Blasticon Biotechnologische Forschung Gmbh Transplantat-Akzeptanz induzierende Zellen monozytären Ursprungs, sowie deren Herstellung und Verwendung
AU2003295471B2 (en) * 2002-11-15 2011-08-25 Genmab A/S Human monoclonal antibodies against CD25
JP2006516957A (ja) 2002-11-15 2006-07-13 セントカー・インコーポレーテツド Ｉｌ−６アンタゴニストの抗血管形成用途
US20040161426A1 (en) 2003-02-04 2004-08-19 Mohit Trikha Use of IL-6 antagonists in combination with steroids to enhance apoptosis
KR101142624B1 (ko) * 2003-02-24 2012-06-15 추가이 세이야쿠 가부시키가이샤 인터루킨-6 길항제를 함유하는 척수손상 치료제
US20040208876A1 (en) 2003-04-18 2004-10-21 Kim Kyung Jin Monoclonal antibodies to hepatocyte growth factor
GB2401040A (en) * 2003-04-28 2004-11-03 Chugai Pharmaceutical Co Ltd Method for treating interleukin-6 related diseases
AU2004268614C1 (en) * 2003-08-27 2010-10-28 Ophthotech Corporation Combination therapy for the treatment of ocular neovascular disorders
CN1874991A (zh) * 2003-08-29 2006-12-06 小野药品工业株式会社 能够结合s1p受体的化合物及其药物用途
JP4960096B2 (ja) 2003-09-22 2012-06-27 ペントラコール ゲゼルシャフト ミット ベシュレンクテル ハフツング Ｉｌ−６の生物学的効果を減じる化合物の使用
JPWO2005027970A1 (ja) * 2003-09-24 2007-11-15 協和醗酵工業株式会社 癌治療用医薬
BRPI0415505A (pt) * 2003-10-17 2006-12-12 Chugai Pharmaceutical Co Ltd agente terapêutico para mesotelioma
KR100556660B1 (ko) 2003-11-11 2006-03-10 국립암센터 Ｈｇｆ의 중화가능 에피토프 및 이에 결합하는 중화 항체
AR048210A1 (es) 2003-12-19 2006-04-12 Chugai Pharmaceutical Co Ltd Un agente preventivo para la vasculitis.
JP4609877B2 (ja) 2004-03-30 2011-01-12 塩野義製薬株式会社 慢性拒絶反応抑制剤
PL1772145T3 (pl) 2004-07-16 2011-08-31 Kyorin Seiyaku Kk Sposób skutecznego stosowania leku i sposób dotyczący zapobiegania efektom ubocznym
AR051071A1 (es) * 2004-08-05 2006-12-20 Wyeth Corp Antagonismo de la actividad del receptor de interleuquina-21
US7884196B2 (en) * 2004-10-06 2011-02-08 Oliver Lawless Vaccine composition comprising methylated DNA and immunomodulatory motifs
EP3026063A1 (fr) 2004-12-28 2016-06-01 Innate Pharma S.A. Anticorps monoclonaux contre nkg2a
WO2006072954A2 (fr) * 2005-01-05 2006-07-13 Compugen Ltd. Nouveaux polynucleotides il-6 codant pour des polypeptides il-6 variants et leurs methodes d'utilisation
PE20061324A1 (es) 2005-04-29 2007-01-15 Centocor Inc Anticuerpos anti-il-6, composiciones, metodos y usos
WO2007043641A1 (fr) 2005-10-14 2007-04-19 Fukuoka University Inhibiteur de dysfonctionnement d'îlots transplantés dans un transplant d'îlots
AU2006305119B2 (en) * 2005-10-21 2012-12-20 Chugai Seiyaku Kabushiki Kaisha Agents for treating cardiopathy
AR057582A1 (es) 2005-11-15 2007-12-05 Nat Hospital Organization Agentes para suprimir la induccion de linfocitos t citotoxicos
WO2007061029A1 (fr) 2005-11-25 2007-05-31 Keio University Agent thérapeutique pour le cancer de la prostate
AR057227A1 (es) 2005-12-09 2007-11-21 Centocor Inc Metodo para usar antagonistas de il6 con inhibidores del proteasoma
ES2382164T3 (es) 2005-12-30 2012-06-05 Merck Patent Gmbh Anticuerpos anti-IL-6 que impiden la unión de la IL-6 en complejo con el IL-6R( ) a la GP130
CN101370521A (zh) * 2006-01-27 2009-02-18 学校法人庆应义塾 伴有脉络膜血管生成的疾病的治疗药
CA2648644C (fr) * 2006-04-07 2016-01-05 Osaka University Promoteur de regeneration musculaire
MX2008014804A (es) * 2006-06-02 2009-01-27 Regeneron Pharma Anticuerpos de afinidad elevada a receptor de il-6 humano.
WO2008020079A1 (fr) 2006-08-18 2008-02-21 Ablynx N.V. Séquences d'acides aminés dirigées contre l'il-6r et polypeptides les contenant utilisés pour le traitement de maladies et de troubles associés au signal médié par il-6
US20080081041A1 (en) * 2006-09-29 2008-04-03 Jeffrey Nemeth Method of Using IL6 Antagonists with Mitoxantrone for Prostate Cancer
CN101646459B (zh) 2007-01-23 2014-02-12 国立大学法人信州大学 慢性排斥反应抑制剂
PT3187506T (pt) 2007-05-21 2019-04-24 Alderbio Holdings Llc Anticorpos contra il-6 e sua utilização
WO2009010539A2 (fr) 2007-07-19 2009-01-22 Ablynx. N.V. Recepteur de l'interleukine-6 (il-6) derive de macaca fascicularis
AR068564A1 (es) 2007-09-26 2009-11-18 Chugai Pharmaceutical Co Ltd Anticuerpo anti- receptor de il-6 (interleuquina 6)
MX2010003450A (es) * 2007-09-26 2010-04-27 Chugai Pharmaceutical Co Ltd Region constante de anticuerpo modificada.
KR102269708B1 (ko) 2008-04-11 2021-06-25 추가이 세이야쿠 가부시키가이샤 복수 분자의 항원에 반복 결합하는 항원 결합 분자
TWI528973B (zh) 2008-06-05 2016-04-11 Chugai Pharmaceutical Co Ltd Nerve infiltration inhibitor
JP2010258402A (ja) 2008-09-26 2010-11-11 Sony Corp 静電容量素子及び共振回路
TWI440469B (zh) 2008-09-26 2014-06-11 Chugai Pharmaceutical Co Ltd Improved antibody molecules
US8323649B2 (en) 2008-11-25 2012-12-04 Alderbio Holdings Llc Antibodies to IL-6 and use thereof
RU2524152C2 (ru) 2009-03-19 2014-07-27 Чугаи Сейяку Кабусики Кайся Средство для лечения ревматоидного артрита
WO2011013786A1 (fr) 2009-07-31 2011-02-03 Maeda Shin Inhibiteur de métastases cancéreuses
WO2011149046A1 (fr) 2010-05-28 2011-12-01 独立行政法人国立がん研究センター Agent thérapeutique contre le cancer du pancréas
RS63800B1 (sr) * 2010-05-28 2022-12-30 Chugai Pharmaceutical Co Ltd Poboljšanje antitumorskog odgovora t ćelije
US8835576B2 (en) 2010-07-09 2014-09-16 Univation Technologies, Llc Ethylene expansion for low temperature refrigeration in polyethylene vent recovery
JPWO2012063875A1 (ja) 2010-11-11 2014-05-12 シスメックス株式会社 ヒト濾胞ヘルパーｔ細胞検出用マーカーおよびヒト濾胞ヘルパーｔ細胞の検出方法
BR112013021725A2 (pt) 2011-02-28 2016-11-01 Genentech Inc marcadores biológicos e métodos para prever resposta aos antagonistas de células b
WO2014200018A1 (fr) 2013-06-11 2014-12-18 独立行政法人 国立精神・神経医療研究センター Procédé permettant de prédire le pronostic post-thérapie d'un patient atteint de sclérose en plaques à périodes progressives et rémittentes (spppr) et procédé permettant de déterminer l'applicabilité d'une nouvelle thérapie
ES2888801T3 (es) 2015-05-19 2022-01-07 Nat Center Neurology & Psychiatry Método para determinar la aplicación de una terapia nueva en pacientes con esclerosis múltiple (EM)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11851486B2 (en) 2017-05-02 2023-12-26 National Center Of Neurology And Psychiatry Method for predicting and evaluating therapeutic effect in diseases related to IL-6 and neutrophils

Also Published As

Publication number Publication date
JPWO2009148148A1 (ja) 2011-11-04
TW201503898A (zh) 2015-02-01
TW201006491A (en) 2010-02-16
CA2728243C (fr) 2020-03-10
CA2728243A1 (fr) 2009-12-10
WO2009148148A1 (fr) 2009-12-10
KR101665729B1 (ko) 2016-10-12
CN102256623A (zh) 2011-11-23
US10717781B2 (en) 2020-07-21
TWI528973B (zh) 2016-04-11
EP2305306A1 (fr) 2011-04-06
EP2305306A4 (fr) 2012-08-29
JP5544290B2 (ja) 2014-07-09
KR20110046399A (ko) 2011-05-04
HK1214514A1 (zh) 2016-07-29
CN104906581A (zh) 2015-09-16
US20110150869A1 (en) 2011-06-23

Similar Documents

Publication Publication Date Title
EP2305306B1 (fr) Inhibiteur de neuro-invasion
US8945558B2 (en) Methods for treating myocardial infarction comprising administering an IL-6 inhibitor
US8529895B2 (en) Method for suppressing the development of graft-versus-host-disease by administering interleukin 6 receptor antibodies
EP1967209B1 (fr) Agent thérapeutique pour le cancer de la prostate
EP2578231B1 (fr) Agent améliorant la réponse de lymphocytes t anti-tumoraux
EP2174667B1 (fr) Agent pour le traitement de l'ophtalmie contenant un inhibiteur du récepteur de l'interleukine 6 en tant qu'ingrédient actif
EP2578233B1 (fr) Agent thérapeutique contre le cancer du pancréas
EP2460538A1 (fr) Inhibiteur de métastases cancéreuses

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20110104

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA RS

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20120731

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 43/00 20060101ALI20120723BHEP

Ipc: A61P 25/00 20060101ALI20120723BHEP

Ipc: A61K 39/395 20060101ALI20120723BHEP

Ipc: C07K 16/28 20060101ALI20120723BHEP

Ipc: A61P 35/00 20060101ALI20120723BHEP

Ipc: A61P 1/18 20060101ALI20120723BHEP

Ipc: A61K 45/00 20060101AFI20120723BHEP

17Q First examination report despatched

Effective date: 20141219

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20150724

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 774320

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160215

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602009036206

Country of ref document: DE

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20160210

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 8

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 774320

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160210

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160510

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160511

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160613

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160610

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602009036206

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

26N No opposition filed

Effective date: 20161111

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160510

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160630

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160630

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160605

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 9

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20090605

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 10

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160605

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160210

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20160630

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230424

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 20230627

Year of fee payment: 15

Ref country code: DE

Payment date: 20230620

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20230622

Year of fee payment: 15